This invention relates generally to radioimmunoassay techniques and more particularly to assay kits for the assay of adenosine-3',5'-cyclic monophosphate and/or guanosine-3',5'-cyclic monophosphate by a radioimmunoassay method and to a method of using the assay kits.
In recent years, adenosine-3',5'-cyclic monophosphate (hereinafter referred to by the abbreviation cAMP) has been widely studied as a mediator of hormone action, and with respect to guanosine-3',5'-cyclic monophosphate (hereinafter referred to by the abbreviation cGMP), also, its physiologic action is being clarified. Particularly from the observation that the contents of cAMP and cGMP within a living body are apt to change when that living body is in an unphysilogic or pathologic state such as a diseased state, the great importance of the assay of the contents of these cyclic nucleotide within a living body not only in basic medical research but also in diagonosis, prevention, and treatment of diseases in the field of clinical medicine is being recognized.
For example, the assays of cAMP and cGMP in samples taken from living bodies such as leucocytes from asthmatic patients, skin of psoriasis patients, blood platelets of thrombocytosis patients, blood and urine of pseudohypoparathyroidism patients, blood and urine of muscular dystrophy patients, and cerebrosphinal fluid of manic-depressive patients are effective in the diagnosis and treatment of these diseases. Furthermore, the clarification of the interrelationships between the results of these assays and numerous diseases is expected hereafter.
However, the methods known heretofore for the assay of cAMP and cGMP cannot be said to be satisfactory on the point of sensitivity and, furthermore, have been accompanied by problems of time and economy in the preparation of samples from organisms. For instance, a nonspecific factor(s) which hinder(s) the assay is present in a sample from an organism, and in order to remove this effect, it is considered necessary to dilute the sample to a high dilution ratio. This dilution, however, further lowers the concentration of the cAMP and cGMP which are present in minute quantities and therefore difficult to assay from the beginning. As a result, this further lowered concentration becomes less than the lower limit of measurability, whereby quantitative measurements are rendered even more difficult.
Furthermore, since there is originally a great difference between the contents within an organism of cAMP and cGMP, a single sample preparation cannot be used as a common sample. In this connection, the compositions and minimum limiting quantities for measuring of cAMP and cGMP assay kits presently sold on the market are as set forth in Tables 1 and 2.
Table 1. ______________________________________ cAMP assay kits on the market Min.Limiting quantity for measuring Product Composition (p mol.*/tube) ______________________________________ .circle.1 Tris/EDTA buffer .circle.2 cAMP standard solution A .circle.3 Tritium-labeled cAMP 0.2 .circle.4 Binding protein .circle.5 Material for separation .circle.1 cAMP standard solution B .circle.2 Iodine-labeled cAMP 0.05 .circle.3 cAMP antibody .circle.1 cAMP standard solution .circle.2 Iodine-labeled cAMP C 1 .circle.3 cAMP antibody .circle.4 Antibody for separation .circle.1 Acetate buffer .circle.2 cAMP standard solution .circle.3 Tritium-labeled cAMP D 1 .circle.4 Binding protein .circle.5 Separation filter .circle.6 Buffer for washing filter ______________________________________ *picomol.
Table 2. ______________________________________ cGMP assay kits on the market Min.limiting quantity for measuring Product Composition (p mol./tube) ______________________________________ .circle.1 cGMP standard solution E .circle.2 Iodine-labeled cGMP 0.05 .circle.3 cGMP antibody .circle.1 cGMP standard solution .circle.2 Iodine-labeled cGMP F .circle.3 cGMP antibody 0.05 .circle.4 Antibody for separation .circle.1 Acetate buffer .circle.2 Tritium-labeled cGMP G .circle.3 cGMP standard solution 0.2 .circle.4 Binding protein ______________________________________
Thus, the minimum limiting quantities for measuring of cAMP and cGMP by means of these kits on the market are 0.05 p mol./tube or higher in all cases, whereby it is not possible by using these kits to achieve the object of ultramicro-assay of cAMP and cGMP which will inevitably become a requirement hereafter.
One known method for improving the measurement sensitivity of radioimmunoassay is the method wherein cAMP is subjected to succinylation thereby to increase the affinity of the cAMP for its antibody, as proposed in Analytical Biochemistry, Volume 56, p.394 through p.407, (1973). In this method, a process (A) in an organic solvent system which comprises dissolving a freeze-dried test specimen of a sample of an organism and 4-morpholino-N,N'-dicyclohexylcarboxamidine in pyridine and adding to the resulting solution a solution of succinic anhydride in dioxane thereby to carry out a succinylation reaction and a process (B) in an aqueous solution system which comprises adding succinic anhydride powder and triethylamine to an aqueous solution of the test specimen thereby to carry out a succinylation reaction are proposed.
The process (A), however, is not practical because of difficulties such as the condition that the prepared form of the test specimen must be freeze-dried and the low efficiency of succinylation due to low solubility of the cAMP in the solvent. In the process (B), since the succinic anhydride is supplied in powder form, and also in consideration of the hydrolyzable nature of succinic anhydride in an aqueous solution system, it is essential to promote instantaneously the dissolution and diffusion of the succinic anhydride into the aqueous solution of the test specimen by a vigorous shaking action in order to cause the succinylation reaction to progress with high efficiency. Accordingly, in addition to the difficulty of handling the powder, equipment for this shaking action is necessary. The simultaneous assay of a large number of test specimens is particularly difficult by this process, which is unsuitable for practice by a simple assay kit. By this process, furthermore, dialysis is used as an expedient for separating the free cAMP and the cAMP bound with the antibody after the antigen-antibody reaction, but special equipment is necessary for the dialysis, and this process has not been satisfactory as a general assay process.